High throughput screening assays using fatty acid synthetic enzymes

ABSTRACT

High throughput screening assays for the identification of potential therapeutic agents able to modulate the activity of enzymes of fatty acid biosynthesis, especially desaturases and/or elongases, are disclosed along with therapeutic uses of the agents identified by such assays for the prevention and/or treatment of diseases related to fatty acid metabolism. Substrates useful in such assays are also described.

[0001] This application claims priority of U.S. Provisional ApplicationSerial No. 60/315,763, filed 29 Aug. 2001, the disclosure of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to high throughput screening assaysfor the identification of potential therapeutic agents and to uses ofagents identified by such assays for the prevention and/or treatment ofdiseases related to fatty acid metabolism.

BACKGROUND OF THE INVENTION

[0003] Acyl desaturase enzymes catalyze the formation of double bonds infatty acids derived from either dietary sources or de novo synthesis inthe liver. Mammals synthesize at least three distinct desaturases, whichcatalyze desaturation fatty acids at different positions. These enzymesare referred to by the carbon number at which the double bond isinserted, which may occur at the Δ9, Δ6 and Δ5 positions. The resultingmono-unsaturated or polyunsaturated fatty acids are substrates forincorporation into phospholipids, triglycerides, and cholesterol esters.

[0004] In human and other mammalian cells, microsomal Δ6 desaturation ofthe essential fatty acids linoleic acid (18:2n-6) and alpha-linolenicacid (18:3n-3) is an initial and rate limiting step in the biosynthesisof both n-6 and n-3 polyunsaturated fatty acids (PUFAs). The microsomalfraction is the fraction, comprising microspheres and other structures,produced by break-up of the rough endoplasmic reticulum followingultracentrifugation and represents a readily producible form of a numberof key cellular enzymes. The products of the aforementioned reactionsare gammalinolenic (18:3n-6) and stearidonic acids (18:4n-3) which aresubsequently elongated to dihomogammalinolenic acid (DGLA) (20:3n-6) andto the 20:4n-3 product, respectively. The resulting fatty acids areutilized as substrates of a Δ5 desaturase that generates arachidonicacid (20:4n-6) and 20:5n-3. The latter are then further elongated to22:4n-6 and 22:5n-3, respectively, and finally used to form the products24:4n-6 and 24:5n-3. The delta-5 desaturase that produces 20:3n9 (orMead acid) may not be the same enzyme that produces arachidonic acidfrom DGLA.

[0005] In addition to the foregoing, a second microsomal Δ6 desaturationoccurs on the PUFAs. The products of this desaturation, 24:5n-6 and24:6n-3, are converted to 22:5n-6 and 22:6n-3, respectively, byperoxisomal β-oxidation (Sprecher, H. (2000) Biochim. Biophys. Acta.1486, 219-231). In addition, two sequential elongations of 20:4n-3(i.e., 22:4n-3 and 24:4n-3) were described by Sauerwald et al. as partof putative alternative steps in the synthesis of 22:6n-3 (Sauerwald, T.U., Hachey, D. L., Jensen, C. L., Chen, H., Anderson, R. E. and Heird W.C. (1997) Pediatr. Res. 41,183-187). These authors proposed that 24:4n-3could undergo Δ9 desaturation to 24:5n-3 which can act as substrate ofthe Δ6 desaturase. Direct elongations on 18:2n-6, 18:3n-3 and 20:3n-6are also reported.

[0006] Evidence shows that the Δ6 desaturase, which recognizes 18-carbonunsaturated fatty acids (18:2n-6 or 18:3n-3), is the same enzyme thatdesaturates 24-carbon substrates (De Antueno, R. J., Knickle, L C,Smith, H., Elliot, M. L., Allen, S. J., Nwaka, S., and Winther, M. D.(2001) FEBS Letters 509, 77-80, and Innis, S. M., Sprecher, H., Hachey,D., Edmond, J. and Anderson, R. E. (1999) Lipids. 34, 139-149. Cho andcoworkers were the first to clone this human Δ6 desaturase and test itsactivity on the generally used 18-carbon PUFA substrate. (Cho, H. P.,Nakamura, M. T. and Clarke, S. D. (1999) J. Biol. Chem. 274, 471-477).In addition, Sauerwald and coworkers (1997) have suggested that two Δ6desaturation steps active on 18:3n-3 and 18:2n-6 would compete not onlywith each other but also with 24:5n-3 and 24:4n-6. Sprecher, in a recentreview addressing this subject, has emphasized that the control of theΔ6 desaturase would be of considerable interest in animals or humanstudies if a single enzyme is active on 4 different fatty acids fromboth n-6 and n-3 families (See: Sprecher, supra).

[0007] The delta-6 desaturase carries out desaturation on at least 4distinct substrates: 18:3n6, 24:4n6, 18:3n3 and 24:5n3. See: De AntuenoR J, Knickle L C, Smith H, Elliott M L, Allen S J, Nwaka S, Winther M D, “Activity of human Delta5 and Delta6 desaturases on multiple n-3 andn-6 polyunsaturated fatty acid,” FEBS Lett. Nov 30;509(1):77-80 (2001);D'andrea S, Guillou H, Jan S, Catheline D, Thibault J N, Bouriel M,Rioux V, Legrand P., “The same rat Delta6-desaturase not only acts on18- but also on 24-carbon fatty acids in very-long-chain polyunsaturatedfatty acid biosynthesis,” Biochem J 364(Pt1):49-55 (May 15, 2002). Inaddition, 18:1 n9 (oleic acid) should also be a substrate (although,perhaps, with lower affinity).

[0008] There are several different elongases present in mammalian cells,which are active on different fatty acid substrates. Some of these havebeen characterized (see Winther et al, WO 02/44320). In at least oneexample a genetic defect in a human elongase is associated with a humandisease, indicating the importance of this enzyme family as atherapeutic target (Zhang, K., Kniazeva, M.,Han, M. et al (2001) NatureGenetics 27, 89-93).

[0009] It has been shown (see Brownlie et al, WO 01/62954) that delta-9desaturase activity may be indirectly measured in a microsomal assay bymeasuring the release of tritium from the C9 and C10 positions ofstearoyl-CoA, in the form of water. In accordance with the presentinvention, this kind of assay is applicable to a wide range of otherdesaturase and elongase enzymes using an efficient and economicaltritium based assay. Using the process disclosed herein, modulators offatty acid or triglyceride metabolism are readily identified.

BRIEF SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention relates to a process foridentifying a desaturase-modulating agent, comprising:

[0011] a) contacting a compound with a microsome, said microsomecomprising a desaturase enzyme activity, in the presence of a substratecomprising a tritium label wherein said label is bound to a desaturasereactive carbon atom and under conditions promoting the desaturation ofsaid substrate at said reactive carbon atom to form a double bond withsaid reactive carbon atom with release of tritiated water,

[0012] b) detecting production of said tritiated water,

[0013] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent thereby identifying a modulator of desaturase activity.

[0014] In specific embodiments thereof, the modulation may be anincrease or a decrease in desaturase activity and may be determined aseither a difference in the amount of said tritiated water detectedand/or a difference in the rate of production of said tritiated water.In addition, the tritium label may be present on one or both of thedesaturase reactive carbon atoms (i.e., the carbon atoms forming thedouble bond resulting from this desaturation reaction). In preferredembodiments, the desaturase is a delta-5 desaturase or a delta-5desaturase.

[0015] Preferred substrates for delta-5 desaturases include 20 carbonbackbone fatty acids, such as a coenzyme A conjugate ofdihomogammalinolenic acid (20:3n6, DGLA), 20:4n3 and 20:2n9, especiallywhere the tritium label is on carbon atom 5 and 6 of the substrate.

[0016] Preferred substrates for the delta-6 desaturase include asubstrate that is a coenzyme A conjugate of a fatty acid having abackbone of 18 carbon atoms or 24 carbon atoms in length, preferably acoenzyme A conjugate of α-linolenic acid (18:3n3), 24:5n3, linoleic acid(18:2n6), 18:1n9 or 24:4n6, especially where the tritium label is oncarbon atom 6 and 7 of the substrate.

[0017] In another aspect, the present invention relates to a process foridentifying an elongase- and/or desaturase-modulating agent, comprising:

[0018] a) contacting a compound with a microsome, said microsome havinga desaturase enzyme activity, in the presence of an elongase and asubstrate of said elongase, wherein said substrate is not a substrate ofsaid desaturase enzyme activity, under conditions promoting elongationof said substrate to form an elongated product wherein said product is asubstrate of said desaturase and comprises a tritium label bound to adesaturase reactive carbon atom and under conditions promotingdesaturation of said substrate of said desaturase to form a double bondinvolving said desaturase reactive carbon atom with release of tritiatedwater,

[0019] b) detecting production of said tritiated water,

[0020] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent,

[0021] thereby identifying a modulator of elongase and/or desaturaseactivity.

[0022] Preferred embodiments include wherein said modulation is adecrease or increase in desaturase and/or elongase activity, preferablywhere the desaturase is a delta-5 desaturase and where the substrate forthe elongase is a 16, 18, 20 or 22-carbon atom backbone fatty acid.

[0023] Substrates of the elongase may also be substrates of the n9pathway (which starts with oleic acid 18:1n9) and the n7 pathway (whichstarts with palmitic acid (16:0). Elongation substrates would be:18:2n9, 20:3n9, 18:1n9, 20:1n9, 22:1n9, 16:0, 16:1n7, 18:1n7, 20:1n7.

[0024] In a further aspect, the present invention relates to a processfor identifying an elongase-modulating agent, comprising:

[0025] a) contacting a compound with a microsome, said microsome havinga desaturase enzyme activity, in the presence of an elongase and asubstrate of said elongase, wherein said compound is not a modulator ofsaid desaturase activity using the process disclosed herein without theelongase for determining modulators of desaturase activity and whereinsaid substrate is not a substrate of said desaturase enzyme activity,under conditions promoting elongation of said substrate to form anelongated product wherein said product is a substrate of said desaturaseand comprises a tritium label bound to a desaturase reactive carbon atomand under conditions promoting desaturation of said substrate of saiddesaturase to form a double bond involving said desaturase reactivecarbon atom with release of tritiated water,

[0026] b) detecting production of said tritiated water,

[0027] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent, and

[0028] thereby identifying a modulator of elongase activity.

[0029] Preferred embodiments include wherein said modulation is adecrease or increase in desaturase and/or elongase activity, preferablywhere the desaturase is a delta-5 desaturase and where the substrate forthe elongase is an 18, 20 or 22-carbon atom backbone fatty acid.

[0030] Substrates of the elongase again would include substrates of then9 pathway (which starts with oleic acid 18:1n9) and the n7 pathway(which starts with palmitic acid (16:0). Elongation substrates would be:18:2n9, 20:3n9, 18:1n9, 20:1n9, 22:1n9,16:0, 16:1n7, 18:1n7, 20:1n7.

[0031] In an additional aspect, the present invention relates to aprocess for identifying a therapeutic agent useful in modifying fattyacid levels to bring about therapeutic interventions in a range ofdiseases, comprising identifying an agent according to themodulator-determining processes of the invention and further comprisingadministering said agent to an animal and then detecting a decrease insaid triglyceride levels following said administration therebyidentifying such a therapeutic agent. Preferred embodiments thereofinclude where the animal is afflicted with metabolic disorders withaltered fatty acid metabolism, most preferably where said animal is ahuman being.

[0032] In a still further aspect, the present invention relates to aprocess for treating a disease of fatty acid metabolism in an animalcomprising administering to an animal afflicted therewith atherapeutically effective amount of a compound first identified as adesaturase and/or elongase modulating agent using an assay as disclosedherein according to the invention, especially where said animal is ahuman being.

[0033] In a yet further aspect, the present invention relates to aprocess for protecting an animal against a disorder involving fatty acidmetabolism comprising administering to an animal predisposed todeveloping such a disorder a therapeutically effective amount of acompound first identified as a desaturase and/or elongase modulatingagent using an assay of the invention, preferably where said animal is ahuman being, most preferably a human genetically predisposed to developsaid disorder.

[0034] In a yet still further aspect, the present invention relates to amethod for producing a product comprising identifying an agent accordingto any of the assay processes of the invention wherein said product isthe data collected with respect to said agent as a result of saidprocess and wherein said data is sufficient to convey the chemicalstructure and/or properties of said agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 sets out the nomenclature employed herein to describespecific fatty acids.

[0036]FIG. 2 sets out the steps involved in the biosynthesis of humanN-7 and N-9 Fatty Acids

[0037]FIG. 3 sets out key steps and associated enzymes in humanbiosynthesis of N-3 and N-6 Poly-Unsaturated Fatty Acids (PUFAs).

DETAILED DESCRIPTION OF THE INVENTION

[0038] This invention relates to high throughput screening assays andtheir methods of use in identifying potential therapeutic agents,especially where such assays employ a microsomal source of enzymes, bothdesaturase and elongase enzymes.

[0039] The assays of the invention identify modulators of specific plantor animal desaturases and elongases that are involved in fatty acidsynthesis. All assays measure the release of tritium, in the form ofwater, from fatty acid substrates that have been tritiated at thespecific site of desaturation which is recognized by the specificdesaturase or elongase in question. Each assay is a simple and highlyeffective high throughput assay that are useful for the identificationof modulators, specifically small molecule modulators, of the specificdesaturase or elongase. These modulators and their analogs havepotential therapeutic activity in a wide variety of human diseases, asdescribed below.

[0040] Known enzymes in this pathway include Human Δ5 desaturase(Genbank Accession No. AF226273), Human Δ6 desaturase (Genbank AccessionNos. AF126799) and those listed in Mukerji et al. (WO 00/12720) as wellas the new human elongases are found in “Human Elongase Genes, UsesThereof, and Compounds for Modulating Same”(see Winther et al, WO02/44320), the disclosure of which is hereby incorporated by referencein its entirety.

[0041] It should be well noted that the enzymes useful in the assays ofthe invention have strict substrate specificities. Thus, in accordancewith the present invention, by adding the proper tritiated substrate,modulators of the enzyme that uses that substrate are readilyidentified.

[0042] The presence of an asterisk (*) indicates the presence of atritium label. In all cases the tritium label is located at thepositions where the desaturation reaction takes place. Table 1 describesthe substrate specificity enzymes assayed according to the presentinvention.

[0043] Preferred assays of this invention are rat or mouse livermicrosomal assays spiked with the specific tritiated substrate. TABLE 1Enzyme Assay Radio-labeled Ligand Comment Delta-5 For delta-5-desaturasethe Delta-5 works desaturase substrate may be the CoA principally on a20 conjugate of dihomogamma carbon substrate. linolenic acid 20:3n6(8,11,14- eicosatrienoic acid), 20:4n3 or 20:2n9. Positions of labelare: DGLA 20:3n6, 20:4n3 or 20:2n9 ³H on C5* and C6* Elongase Varioussubstrates possible. Example: Since Delta- Example: GLA 18:3n6-³H on 5works principally on C3 and C4 is elongated to substrates with 20 20:3n6which is a substrate for carbons, by adding desaturation by delta-5labeled 18 carbon desaturase. substrate, the Or desaturation only EPA20:5n3 is elongated to occurs to the extent of 22:5 n6 and then to24:5n6, the success of the EPA being labeled on C2 and elongasereaction. C3 for the two elongation Thus modulators of steps. The ³H isreleased when elongase are the 24:5 is desaturated by D6D. identifiable.Delta-6 For delta-6-desaturase the Works on selected 18- desaturasesubstrate may be the CoA C or 24C substrates, conjugate of linoleicacid, but not on 20C 18:2n6 (6,9,12-octadeca - substrates. Thus thedienoic acid) or alpha-linolenic ³H on C6 will not acid 18:3n3 (9,12,15-measure delta 5 octadecatrienoic acid), 24:4n6, activity. 24:5n3 or18:1n9. Linoleic acid 18:2n6 - ³H on C6* and C7* or 24:4n6 - ³H on C6*and C7* or 24:5n3 - ³H on C6* and C7* or 18:3n3 - ³H on C6* and C7*

[0044] In accordance with the foregoing, the present invention relatesto a process for identifying a desaturase-modulating agent, comprising:

[0045] a) contacting a compound with a microsome, said microsomecomprising a desaturase enzyme activity, in the presence of a substratecomprising a tritium label wherein said label is bound to a desaturasereactive carbon atom and under conditions promoting the desaturation ofsaid substrate at said reactive carbon atom to form a double bond withsaid reactive carbon atom with release of tritiated water,

[0046] b) detecting production of said tritiated water,

[0047] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent

[0048] thereby identifying a modulator of desaturase activity.

[0049] In particular embodiments of this process, the modulation may bean increase or a decrease in the desaturase activity and includesembodiments wherein the difference of step (c) is a difference in theamount of said tritiated water detected and/or a difference in the rateof production of said tritiated water. For example, if an inhibitor isan irreversible inhibitor of the enzyme, the reaction may not go tocompletion so that the amount of tritiated water detected following aspecific time period of said reaction is less than where the compound tobe tested is not present. Alternatively, where said compound is acompetitive or non-competitive inhibitor of the desaturase, the reactionkinetics may be temporally different but the overall production oftritiated water may be the same. In addition, the detection of tritiatedwater within the processes of the invention may be qualitative orquantitative so that actual amounts of product may be measured orreaction in the presence of a compound to be tested for modulatingactivity may be compared to cases where the compound is not present.Further, measurements of production of tritiated water may be used tocompare the activity of one test compound against another. For example,should one compound cause a decrease in the rate of production oftritiated water that is twice the decrease produced by a secondcompound, the first compound may be deemed within the invention to betwice as effective in modulating, or inhibiting, the enzyme as thesecond compound. By screening a series of test compounds, the mostadvantageous one can be found.

[0050] A substrate for desaturation will of course be converted to aproduct that contains a carbon-carbon double bond. The carbon atoms inthis substrate that form this double bond as a result of reaction withthe desaturase are referred to herein as the desaturase reactive carbonatoms. It is one, or both, of these carbon atoms that contains thetritium label where tritium replaces one of the hydrogens on theotherwise saturated carbon atom. Thus, in one embodiment of theinvention the tritium label is attached to only one of the reactivecarbon atoms of a substrate molecule while in a separate embodiment thetritium label is attached to two reactive carbon atoms of the samesubstrate molecule. Neither of these embodiments serves to limit theoverall utility of the invention disclosed herein.

[0051] In other specific embodiments of the processes of the presentinvention, the desaturase is a delta-5 desaturase. In a furtherembodiment, the substrate is a coenzyme A conjugate of a fatty acidhaving a backbone of length 20 carbon atoms, such as where thedesaturase is a delta-5 desaturase. In preferred embodiments, thesubstrate of said desaturase is a substrate described herein, such as inTable 1, especially where the substrate is a coenzyme A conjugate of amember selected from the group consisting of dihomogammalinolenic acid,DGLA (20:3 n6), 20:2n9 and 20:4n3. In specific embodiments thereof, thetritium label is on carbon atom 5 and 6 of the substrate.

[0052] In a further embodiment of the processes of the invention, thedesaturase is a delta-6 desaturase. In other embodiments thereof, thesubstrate is a coenzyme A conjugate of a fatty acid having a backbone of18 carbon atoms or 24 carbon atoms in length. In a preferred embodiment,the substrate is one as recited herein, such as one of those of Table 1,preferably where the substrate is a coenzyme A conjugate of a memberselected from the group consisting of linoleic acid (18:2n6),alpha-linolenic acid (18:3n3), oleic acid (18:1 n9), 24:4n6 and 24:5n3.In a highly preferred embodiment thereof, the tritium label is on carbonatom 6 and 7 of the substrate.

[0053] The present invention further relates to a process foridentifying an elongase- and/or desaturase-modulating agent, comprising:

[0054] a) contacting a compound with a microsome, said microsome havinga desaturase enzyme activity, in the presence of an elongase and asubstrate of said elongase, wherein said substrate is not a substrate ofsaid desaturase enzyme activity, under conditions promoting elongationof said substrate to form an elongated product wherein said product is asubstrate of said desaturase and comprises a tritium label bound to adesaturase reactive carbon atom and under conditions promotingdesaturation of said substrate of said desaturase to form a double bondinvolving said desaturase reactive carbon atom with release of tritiatedwater,

[0055] b) detecting production of said tritiated water,

[0056] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent,

[0057] thereby identifying a modulator of elongase and/or desaturaseactivity.

[0058] In a particular embodiment of such process, the modulation is adecrease in desaturase and/or elongase activity or an increase indesaturase and/or elongase activity. In other preferred embodiments,said difference of step (c) is a difference in the amount of saidtritiated water detected or a difference in the rate of production ofsaid tritiated water as described hereinabove and wherein said tritiumlabel is attached to only one desaturase reactive carbon atom of asubstrate molecule or is attached to two desaturase reactive carbonatoms of the same substrate molecule. In a preferred embodiment, thedesaturase is a delta-5 desaturase. In another preferred embodiment, thesubstrate of said elongase comprises a 16, 18, 20 or 22-carbon atombackbone fatty acid, most preferably 18 carbons.

[0059] Substrates of the elongase may also be substrates of the n9pathway (which starts with oleic acid 18:1n9) and the n7 pathway (whichstarts with palmitic acid (16:0). Preferred elongation substrates wouldbe: 18:2n9, 20:3n9, 18:1 n9, 20:1 n9, 22:1 n9, 16:0, 16:1 n7, 18:1 n7,20:1 n7. It should be noted that for the EPA substrate noted in Table 1(and labeled at C2 and C3) the substrate must undergo two sequentialelongations and a delta-6 desaturation to release the tritiated waterproduct. In such an assay, either step could be inhibited by a testcompound but the overall result would be to produce a relatively smallnumber of compounds for further screening.

[0060] A variety of synthetic routes for making the tritiated substratesrequired for this invention are known to those skilled in the art. Anexample is the DGLA substrate employed in the delta-5 desaturasereaction. This 20:3n-6 fatty acid requires a tritium atom on the C5 andC6 carbons (counting from the COOH or CoA head group) in order tomeasure the delta-5 desaturation step to form arachadonic acid (AA). Thetechniques include enzymatic retroconversion of AA-CoA in the presenceof tritiated water and delta-5 desaturase. This reaction simply employesthe reversible enzymatic activity of delta-5 desaturase, and is drivenby excess AA-CoA and also plant, algal or microbe based synthesis in thepresence of tritiated malonyl-CoA. In this reaction, an algae, plantcell or microbe (or an extract thereof) is presented with a C14:0substrate and tritiated malonyl-CoA. Natural enzymes in these organismswill extend the chain to creat C16:0, C18:0, C18:1 and C18:2 substrates.After sufficient time to generate the C16:0, the reaction is floodedwith cold (or un-tritiated) malonyl-CoA. The chain extension continueswith cold malonyl-CoA. The resulting C18:2 product is then exposed to adelta-6 desaturase and an elongase to generate DGLA with a predominanceof tritiums at the C5-C6 carbons. In addition, total synthesis of fattyacids has been explored by several groups. Though yields are low, it ispossible to generate a DGLA from 2 carbon substrates, adding in atritiated pair of carbons at the C5-C6 carbons. It is also possible touse DGLA that is tritiated at all carbons. This substrate iscommercially available. When employed in a microsome extract containingan abundance of delta-5 desaturase activity, the predominant and perhapsexclusive reduction takes place at C5-C6. There may generally be ahigher level of background when using a fully tritiated substrate, butassay conditions can be optimized by those skilled in the art todistinguish delta-5 desaturase activity from other reactions. Usefulsynthetic procedures are also found in U.S. Pat. No. 6,441,035, thedisclosure of which is hereby incorporated by reference in its entirety.

[0061] As used herein, the meaning of “delta-5 desaturase”, “delta-6desaturase” and/or “elongase” in respect to screening assays should betaken to include all functional variants of the underlying genes orproteins (depending on use), including all known or yet to be discoveredalleles, variants, polymorphisms or mutants of such genes/proteins, andespecially including all orthologs and homologs from different speciessuch as the human, mouse or rat forms disclosed previously, forms fromother mammals, other vertebrates, and those forms found in invertebratesand single cell organisms, viruses and the like. Of course, screeningassays need to employ an enzyme which has the required biologicalactivity in order to identify compounds which inhibit that activity.These compounds that are therapeutic agents of the invention, or analogsthereof. In a preferred embodiment, the screening assay employs amammalian form of the gene, such as a microsomal extract of a mammaliancell or a recombinant cell transformed with a plasmid expressionconstruct containing a functional form of the gene. Those skilled in theart know how to identify such variants and orthologs from publicdatabases of gene/protein sequence (i.e. GenBank); or alternatively canprepare microsomal extracts from cells of animals where the exactsequence of the gene/protein is not yet known.

[0062] The present invention has a number of therapeutic uses becausecompounds that modulate enzymes of fatty acid metabolism are useful incontrolling the deleterious effects of a host of diseases caused by, orrelated to, disorders of fatty acid metabolism, such as the manydiseases related to triglyceride metabolism. A non-limiting summary ofdiseases and disorders which are known in the art to relate to the fattyacid metabolism pathways controlled by delta-5 desaturase, delta-6desaturase and elongases (for example, those in FIG. 2 and FIG. 3) andfor which the compounds identified by the screening assays of thisinvention may be used, is: inflammation, pain, analgesia, vasculardisease and cardiovascular disease, dyslipidemia, diabetes (Type I orType II), obesity, metabolic disease, metabolic syndrome, and skindisorders (including skin cancer). These broad categories cover a widerange of specific diseases and disorders known to those in the art.

[0063] The present invention further relates to a process foridentifying an elongase-modulating agent, comprising:

[0064] a) contacting a compound with a microsome, said microsome havinga desaturase enzyme activity, in the presence of an elongase and asubstrate of said elongase, wherein said compound is not a modulator ofsaid desaturase activity using the process of claim 1 and wherein saidsubstrate is not a substrate of said desaturase enzyme activity, underconditions promoting elongation of said substrate to form an elongatedproduct wherein said product is a substrate of said desaturase andcomprises a tritium label bound to a desaturase reactive carbon atom andunder conditions promoting desaturation of said substrate of saiddesaturase to form a double bond involving said desaturase reactivecarbon atom with release of tritiated water,

[0065] b) detecting production of said tritiated water,

[0066] c) detecting a difference in the amount or rate of production ofsaid tritiated water compared to when the compound of step (a) is notpresent, and

[0067] thereby identifying a modulator of elongase activity.

[0068] As described elsewhere herein, embodiments include cases wherethe modulation is a decrease in desaturase and/or elongase activity oris an increase in desaturase and/or elongase activity, wherein saiddifference of step (c) is a difference in the amount of said tritiatedwater detected or a difference in the rate of production of saidtritiated water and wherein said tritium label is attached to only onedesaturase reactive carbon atom of a substrate molecule or is attachedto two desaturase reactive carbon atoms of the same substrate molecule.

[0069] In a preferred embodiment of this process, the desaturase is adelta-5 or delta-6 desaturase. In a highly preferred embodiment, thesubstrate of said elongase comprises a fatty acid having a backbone of18 carbon atoms so that the product has a backbone of 20 carbon atoms(since delta-5 desaturases generally only utilize 20 carbon atom fattyacids as substrates). Of course, this in no way limits the utility ofthe invention for detecting modulators of elongase activity since thesubstrate for the elongase may have any number of carbons, generally aneven number, so long as the desaturase used in the assay accepts assubstrate the corresponding product of the elongase.

[0070] Other substrates of the elongase include 16, 18, 20 and 22 carbonatom backbone fatty acids, especially 18 carbon atoms. Substrates of theelongase may again be substrates of the n9 pathway (which starts witholeic acid 18:1n9) and the n7 pathway (which starts with palmitic acid(16:0), with preferred elongation substrates would be: 18:2n9, 20:3n9,18:1n9, 20:1n9, 22:1n9, 16:0, 16:1n7, 18:1n7, 20:1n7

[0071] Alternatively, such agents can be utilized in the prevention ofonset of such disorders, especially in an animal, preferably a human,predisposed, genetically or otherwise, to develop such disorders at sometime during the life of said animal. Thus, the present invention alsorelates to a process for identifying a therapeutic agent useful inreducing elevated triglyceride levels comprising identifying adesaturase and/or elongase modulating agent according to a process ofthe invention and further comprising administering said agent to ananimal and then detecting a decrease in said triglyceride levelsfollowing said administration thereby identifying such a therapeuticagent. In a preferred embodiment thereof, said animal is an animalafflicted with elevated triglyceride levels. In a highly preferredembodiment, said animal is a human being.

[0072] When used for such therapeutic, or even research, purposes, thecompounds or other agents identified by the assay processes of theinvention will commonly be present, by solution, suspension, orotherwise, in a pharmaceutically acceptable carrier, including anyuseful diluents or excipients. The pharmaceutical compositions usefulherein include any pharmaceutical agent that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Pharmaceutically acceptable carriers include, but are not limited to,liquids such as water, saline, glycerol and ethanol, and the like.Although intravenous administration is preferred, any appropriate routeof administration may be employed, for example, perenteral,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, or oral administration.Therapeutic formulations may be in the form of liquid solutions orsuspension; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

[0073] A thorough discussion of pharmaceutically acceptable carriers,diluents, and other excipients is presented in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for agonists of theinvention include ethylenevinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation may contain excipients, or example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

[0074] The present invention also relates to a process that comprises amethod for producing a product comprising identifying an agent accordingto one of the disclosed processes for identifying such an agent (i.e.,the modulators of desaturase and/or elongase activity disclosed herein,as well as any other modulators of fatty acid metabolism that can beidentified using the assays of the present invention) wherein saidproduct is the data collected with respect to said agent as a result ofsaid identification process, or assay, and wherein said data issufficient to convey the chemical character and/or structure and/orproperties of said agent. For example, the present inventionspecifically contemplates a situation whereby a user of an assay of theinvention may use the assay to screen for compounds having the desiredenzyme modulating activity and, having identified the compound, thenconveys that information (i.e., information as to structure, dosage,etc) to another user who then utilizes the information to reproduce theagent and administer it for therapeutic or research purposes accordingto the invention. For example, the user of the assay (user 1) may screena number of test compounds without knowing the structure or identity ofthe compounds (such as where a number of code numbers are used the firstuser is simply given samples labeled with said code numbers) and, afterperforming the screening process, using one or more assay processes ofthe present invention, then imparts to a second user (user 2), verballyor in writing or some equivalent fashion, sufficient information toidentify the compounds having a particular modulating activity (forexample, the code number with the corresponding results). Thistransmission of information from user 1 to user 2 is specificallycontemplated by the present invention.

[0075] Because the enzymes discussed herein are clinically relevant, thepresent invention also relates to a method for preventing alipid-related disease or condition comprising administering to a patientat risk of developing said disease a therapeutically effective amount ofan agent that has activity in any of the assays disclosed herein, suchas any of the screening procedures of the invention. In a preferredembodiment, the method is one wherein said agent was first identified ashaving such activity using said assay. Thus, any agents that have, orwould have, activity in one or more of the assays of the invention canfind use in preventing such disease or condition.

[0076] As used herein, the term “disease or condition” relatesespecially to maladies as recognized by the medical community as well asconditions that for technical reasons may not commonly be explicitlydefined or recognized as a “disease,” such as the condition of beingoverweight, or which, because a causative agent has not been identifiedor etiology described, other than a defined set of symptoms, isconsidered a syndrome rather than a disease.

[0077] The present invention further relates to a method for treating alipid-related disease or condition comprising administering to a patientafflicted with a lipid-related disease or condition a therapeuticallyeffective amount of an agent that has activity in an assay of theinvention. In a preferred embodiment thereof, said agent was firstidentified as having such activity using said assay.

[0078] The lipid-related disease or condition contemplated by themethods of the invention include any of the diseases of fatty acidmetabolism. In a preferred embodiment of the present invention, thelipid-related disease or condition to be treated and/or prevented, is amember selected from the group consisting of eczema, cardiovascular,inflammation, Sjögren's syndrome, gastrointestinal disorders, a viraldisease, postviral fatigue, a body weight disorder, a psychiatricdisorder, cancer, cystic fibrosis, endometriosis, pre-menstrualsyndrome, alcoholism, congenital liver disease, Alzheimer's syndrome,hypercholesterolemia, autoimmune disorders, atopic disorders, acuterespiratory distress syndrome, articular cartilage degradation, diabetesand a complication of diabetes.

[0079] It should be noted that the disclosures of all publications,including research articles as well as published patent applications,cited herein are deemed to be incorporated by reference in theirentirety.

[0080] In carrying out the procedures of the present invention it is ofcourse to be understood that reference to particular buffers, media,reagents, cells, culture conditions and the like are not intended to belimiting, but are to be read so as to include all related materials thatone of ordinary skill in the art would recognize as being of interest orvalue in the particular context in which that discussion is presented.For example, it is often possible to substitute one buffer system orculture medium for another and still achieve similar, if not identical,results. Those of skill in the art will have sufficient knowledge ofsuch systems and methodologies so as to be able, without undueexperimentation, to make such substitutions as will optimally servetheir purposes in using the methods and procedures disclosed herein.

[0081] The invention is described in more detail in the followingnon-limiting examples. It is to be understood that these methods andexamples in no way limit the invention to the embodiments describedherein and that other embodiments and uses will no doubt suggestthemselves to those skilled in the art.

[0082] In the following example, the term “fatty acid” refers to theparticular fatty acid being utilized as a substrate since the processesof the invention permit a wide range of different substrates to betested depending on the enzyme activity to be determined (delta-5 ordelta-6 desaturase) and the source of tissue. When a fatty acid ischosen it is used for both the tritium label as well as the non-labeledsolutions (reagents 5 and 6 as described in the example). The correctchoice of fatty acid substrate and the position of the tritium label iscritical for the success of the assay. The radio-labeled compoundsdescribed here are entirely novel whether as free fatty acids or whenconjugated to Coenzyme A.

[0083] It should further be noted that multiple elongases exist and thatand the disclosed assays can be used for any of these provided that thecorrect substrate is employed. In addition, a cell or tissue extract canbe used as source where the elongase of interest is amply present.

EXAMPLE 1 Large Volume Method for Delta-5 or Delta-6 Desaturase ActivityAssay In Liver Microsomes

[0084] A. Buffers and Reagents

[0085] 1. 1 M NaH₂PO₄

[0086] add 120 gm NaH₂PO₄ to ˜800 ml water

[0087] bring to 1 L volume with water

[0088] store at room temp.

[0089] 2. 1 M PK buffer

[0090] add 174.18 gm K₂HPO₄ to ˜800 ml water

[0091] adjusted to pH 7.2 with 1 M NaH₂PO₄

[0092] bring to 1 L volume with water

[0093] dilute this solution 10-fold with water to yield 0.1 M PK buffer

[0094] store at room temp.

[0095] 3. 10 mM NaAcetate:50% ethanol solution

[0096] add 82.03 gm NaC₂H₃O₂ to ˜800 ml water

[0097] adjust to pH 5.6 with glacial acetic acid

[0098] bring to 1 L volume with water

[0099] dilute this solution 50-fold to yield 20 mM stock; verify pH 5.6

[0100] add 10 ml 20 mM stock to 10 ml 100% ethanol to yield final 10 mM

[0101] NaC₂H₃O₂/50% ethanol solution

[0102] store at room temp.

[0103] 4. 20 mM NADH (Sigma No. N-8129)

[0104] add 14.213 mg NADH per ml of 0.1 M PK buffer (should be madefresh each day); store stock crystals at −20° C. in desiccator; keep 20mM

[0105] solution on ice until use

[0106] 5. 1.5 mM fatty acid-CoA

[0107] add correct amount of fatty acid to 10 mM NaC₂H₃O₂/50% ethanolsolution

[0108] (should be made fresh each day); store stock crystal at −20° C.;keep 1.5 mM solution on ice until use

[0109] 6. Fatty acid, ³H-labeled, for example as obtained from AmericanRadiolabeled Chemicals. The selection of fatty acid depends on whichenzyme is being assayed.

[0110] For delta-6-desaturase the substrate may be the CoA conjugate oflinoleic acid (6,9,12-octadecadienoic acid) or alpha-linolenic acid(9,12,15-octadecatrienoic acid), 24:4 n6 or 24:5 n3.

[0111] For delta-5-desaturase the substrate may be the CoA conjugate ofdihomogamma linolenic acid (8,11,14-eicosatrienoic acid) or 20:4 n3. Inall cases the tritium label is located at the positions where thedesaturation reaction takes place.

[0112] use 1-2 μCi/assay point

[0113] stock concentration is 1 mCi/ml

[0114] store at −20° C.; keep on ice while pipetting to minimizeevaporation of solvent

[0115] 7. 10×PBS stock

[0116] To 800 ml water add the following:

[0117] 80 gm NaCl

[0118] 2 gm KCl

[0119] 11.5 gm Na₂HPO₄

[0120] 2 gm KH₂PO₄

[0121] adjust pH to 7.4 with 1M NaOH

[0122] bring to 1 L to yield 10×stock

[0123] note: dilute 10×stock 10-fold with distilled water to make 1×PBSmedium

[0124] store at room temp.

[0125] 8. Charcoal solution

[0126] add 100 gm activated charcoal (Sigma No. C-3014) to 1 L 1×PBSmedium

[0127] and store at 4° C.; prior to and during use, this must be stirredconstantly to keep the charcoal in suspension

[0128] 9. 6% PCA solution

[0129] add 100 ml 60% stock solution of perchloric acid to 900 ml water

[0130] store at room temp.

[0131] 10. Liver microsomes

[0132] stock concentration should be 5 mg total protein/ml of 0.1 M PKbuffer and

[0133] stored at −80° C.; freeze-thaw once to minimize loss of enzymeactivity

[0134] In one embodiment, assays may employ a large volume “Bench”method as follows:

[0135] The protocol for the Bench method yields a total volume of 1.1 mlper assay point (0.2 ml reaction, 0.2 ml PCA and 0.7 ml charcoal).

[0136] 1. Make assay pre-mix with following composition (per assaypoint):

[0137] 4 μl 1.5 mM fatty acid-CoA (0.03 mM final)

[0138] 1 μl 1 mCi/mi radioactive ³H fatty acid (1 μCi)

[0139] 20 μl 20 mM NADH (2 mM final)

[0140] 155 μl 0.1 M PK buffer

[0141] To make assay pre-mix for N number of assay points:

[0142] a. multiply reagent volumes by N

[0143] b. multiply resulting products for each reagent by 1.05 to yielda 5% “cushion”(which can be adjusted up or down depending on theaccuracy of liquid handling).

[0144] 2. Add assay pre-mix (180 μl/assay point) to 20 μl of microsomesto initiate reaction

[0145] a. reaction is run at room temp

[0146] b. duration of reaction can range between 5-30 mins

[0147] It is advisable to add “test” compounds to microsomes as apre-incubation period prior to the addition of assay pre-mix.

[0148] 3. Add 100 μl of 6% PCA to quench reaction and vortex the sampleto mix

[0149] 4. Add 700 μl of charcoal suspension to sediment the unusedsubstrate

[0150] 5. Vigorously vortex sample to mix

[0151] 6. Centrifuge sample: 13,000 rpm, 10 minutes, 4° C.

[0152] 7. Remove 400 μl supernatant and transfer to counting vial (andcare must be taken not to collect any of the charcoal pellet or thinfilm remaining on the surface since this contains radioactivesubstrate).

[0153] 8. Add 5 ml of cocktail for liquid scintillation counting (LSC),mix and count the sample

[0154] For the determination of background radiation, a “blank” is usedto determine the background signal which we have defined as the amountof ³H remaining in the supernatant independent of microsomal enzymeactivity. Blanks are samples where PCA is added PRIOR to the addition of180 μl reaction mixture. For newly purchased radioactive fatty acid-CoA,blanks were ˜2% of the maximum signal. An increase in the blank valueand a decrease in the signal magnitude is diagnostic of substratebreakdown during storage.

EXAMPLE 2 Small Volume Method

[0155] Alternatively, assays may utilize a small volume “HTS” method.The protocol for the small volume HTS method yields a total volume of0.21 ml per assay point (0.1 ml reaction, 0.01 ml PCA and 0.1 mlcharcoal). Buffer compositions are provided in Example 1.

[0156] 1. To reduce total volume to 0.21 ml we have made the followingchanges to the assay pre-mix (volumes per assay point):

[0157] 2 μl 1.5 mM fatty acid-CoA (or a 1 μl 1 mCi/ml radioactive ³Hfatty acid-CoA); 10 μl 20 mM NADH; 67 μl 0.1 M PK buffer

[0158] To make assay pre-mix for N number of assay points:

[0159] a. multiply reagent volumes by N

[0160] b. multiply resulting products for each reagent by 1.05 to yielda 5% “cushion”

[0161] c. note: this cushion can be adjusted up or down depending onaccuracy of liquid handling.

[0162] 2. Add assay pre-mix (80 μl/assay point) to 20 μl of microsomesto initiate reaction

[0163] 3. Add 10 μl of 60% PCA and vortex the sample to mix

[0164] 4. Add 100 μl of charcoal suspension

[0165] 5. Vigorously vortex sample to mix

[0166] 6. Centrifuge sample: 13,000 rpm, 10 minutes, 4° C.

[0167] 7. Remove 50 μl supernatant and transfer to counting vial (again,care was taken not to collect any of the charcoal pellet or thin filmremaining on the surface due to the presence of radioactive substratetherein).

[0168] 8. Add cocktail for LSC, mix and count sample

What is claimed is:
 1. A process for identifying a delta-6desaturase-modulating agent, comprising: a) contacting a test compoundwith delta-6 desaturase, in the presence of a substrate comprising atritium label bound to a delta-6 desaturase reactive carbon atom, underconditions promoting the desaturation of said substrate at said reactivecarbon atom to form a double bond with said reactive carbon atom withrelease of tritiated water, b) determining production of said tritiatedwater, and c) determining a difference in the amount or rate ofproduction of said tritiated water compared to when the contacting ofstep (a) does not occur wherein a difference in production of tritiatedwater identifies the test compound as a delta-6 desaturase modulatingagent.
 2. The process of claim 1, wherein said modulation is a decreasein desaturase activity.
 3. The process of claim 1, wherein saidmodulation is an increase in desaturase activity.
 4. The process ofclaim 1, wherein said difference of step (c) is a difference in theamount of said tritiated water detected.
 5. The process of claim 1,wherein said difference of step (c) is a difference in the rate ofproduction of said tritiated water.
 6. The process of claim 1, whereinsaid tritium label is attached to only one of the reactive carbon atomsof a substrate molecule.
 7. The process of claim 1, wherein said tritiumlabel is attached to two reactive carbon atoms of the same substratemolecule.
 8. The process of claim 1, wherein said substrate is acoenzyme A conjugate of a fatty acid having a backbone of 18 carbonatoms or 24 carbon atoms in length.
 9. The process of claim 1, whereinsaid substrate is a coenzyme A conjugate of a member selected from thegroup consisting of linoleic acid (18:2n6), 24:4n6, alpha-linolenic acid(18:3n3), 24:5n3 and 18:1n9.
 10. The process of claim 9, wherein saidtritium label is on carbon atom 6 and 7 of the substrate.
 11. Theprocess of claim 1 wherein said delta-6 desaturase is part of a wholecell.
 12. The process of claim 1 wherein said delta-6 desaturase is partof a microsomal extract or semi-purified cell extract.
 13. The processof claim 1 wherein said delta-6 desaturase is a purified delta-6desaturase protein.
 14. A process for identifying a therapeutic agentuseful in modifying fatty acid levels comprising identifying an agentaccording to the process of claim 1, and further comprisingadministering said agent to an animal and then detecting a change infatty acid levels following said administration thereby identifying sucha therapeutic agent.
 15. The process of claim 14 wherein said animal isan animal afflicted with a metabolic disorder resulting in altered fattyacid levels, or where altering fatty acid levels brings about atherapeutic benefit.
 16. The process of claim 14 wherein said animal isa human being.
 17. A process for treating a disease of fatty acidmetabolism in an animal comprising administering to an animal afflictedtherewith a therapeutically effective amount of a compound firstidentified as a delta-6 desaturase modulating agent using an assay ofclaim
 1. 18. The process of claim 17 wherein said animal is a humanbeing.
 19. A process for protecting an animal against a disorderinvolving fatty acid metabolism comprising administering to an animalpredisposed to developing such a disorder a therapeutically effectiveamount of a compound first identified as a delta-6 desaturase-modulatingagent according to the process of claim
 1. 20. The process of claim 19wherein said animal is a human being.
 21. The process of claim 19wherein said animal is genetically predisposed to develop said disorder.22. A method for producing a product comprising identifying an agentaccording to the process of claim 1 wherein said product is the datacollected with respect to said agent as a result of said process andwherein said data is sufficient to convey the chemical structure and/orproperties of said agent.
 23. A method for preventing a lipid-relateddisease or condition comprising administering to a patient at risk ofdeveloping said disease a therapeutically effective amount of an agentthat has activity according to the process of claim
 1. 24. The method ofclaim 23 wherein said agent was first identified as having such activityusing said assay.
 25. The method of claim 24 wherein said disease orcondition is a member selected from the group consisting of eczema,cardiovascular, inflammation, Sjögren's syndrome, gastrointestinaldisorders, a viral disease, postviral fatigue, a body weight disorder, apsychiatric disorder, cancer, cystic fibrosis, endometriosis,pre-menstrual syndrome, alcoholism, congenital liver disease,Alzheimer's syndrome, hypercholesterolemia, autoimmune disorders, atopicdisorders, acute respiratory distress syndrome, articular cartilagedegradation, diabetes and a complication of diabetes.
 26. A method fortreating a lipid-related disease or condition comprising administeringto a patient afflicted with a lipid-related disease or condition atherapeutically effective amount of an agent that has activity accordingto the process of claim
 1. 27. The method of claim 26 wherein said agentwas first identified as having such activity using said assay.
 28. Themethod of claim 26 wherein said disease or condition is a memberselected from the group consisting of eczema, cardiovascular,inflammation, Sjögren's syndrome, gastrointestinal disorders, a viraldisease, postviral fatigue, a body weight disorder, a psychiatricdisorder, cancer, cystic fibrosis, endometriosis, pre-menstrualsyndrome, alcoholism, congenital liver disease, Alzheimer's syndrome,hypercholesterolemia, autoimmune disorders, atopic disorders, acuterespiratory distress syndrome, articular cartilage degradation, diabetesand a complication of diabetes.